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    Of General Direct Cloning of POR: Construction T-Vectors, A Rapid and System For Unmodified Products

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    alejandro
    This document describes a new method for cloning unmodified PCR products using T-vectors. The method exploits the template-independent terminal transferase activity of Taq polymerase, which adds a single nucleotide to the 3' end of PCR products, usually an adenosine. The researchers digest a plasmid with EcoRV and incubate it with Taq polymerase and dTTP, adding a single thymidine to the 3' ends of the linearized plasmid. This creates complementary overhangs between the vector and PCR product that facilitates efficient ligation without additional enzymatic processing of the PCR product. The researchers were able to clone nine different PCR products into the T-vector on the first attempt using this simple and general method.

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    Of General Direct Cloning of POR: Construction T-Vectors, A Rapid and System For Unmodified Products

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    alejandro
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    This document describes a new method for cloning unmodified PCR products using T-vectors. The method exploits the template-independent terminal transferase activity of Taq polymerase, which adds a single nucleotide to the 3' end of PCR products, usually an adenosine. The researchers digest a plasmid with EcoRV and incubate it with Taq polymerase and dTTP, adding a single thymidine to the 3' ends of the linearized plasmid. This creates complementary overhangs between the vector and PCR product that facilitates efficient ligation without additional enzymatic processing of the PCR product. The researchers were able to clone nine different PCR products into the T-vector on the first attempt using this simple and general method.

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    nar00241-0174

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    This document describes a new method for cloning unmodified PCR products using T-vectors. The method exploits the template-independent terminal transferase activity of Taq polymerase, which adds a single nucleotide to the 3' end of PCR products, usually an adenosine. The researchers digest a plasmid with EcoRV and incubate it with Taq polymerase and dTTP, adding a single thymidine to the 3' ends of the linearized plasmid. This creates complementary overhangs between the vector and PCR product that facilitates efficient ligation without additional enzymatic processing of the PCR product. The researchers were able to clone nine different PCR products into the T-vector on the first attempt using this simple and general method.

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    © All Rights Reserved
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    25 views1 page

    Of General Direct Cloning of POR: Construction T-Vectors, A Rapid and System For Unmodified Products

    Uploaded by

    alejandro
    This document describes a new method for cloning unmodified PCR products using T-vectors. The method exploits the template-independent terminal transferase activity of Taq polymerase, which adds a single nucleotide to the 3' end of PCR products, usually an adenosine. The researchers digest a plasmid with EcoRV and incubate it with Taq polymerase and dTTP, adding a single thymidine to the 3' ends of the linearized plasmid. This creates complementary overhangs between the vector and PCR product that facilitates efficient ligation without additional enzymatic processing of the PCR product. The researchers were able to clone nine different PCR products into the T-vector on the first attempt using this simple and general method.

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    of 1

    1154 Nucleic Acids Research, Vol. 19, No.

    Construction of T-vectors, a rapid and general system for


    direct cloning of unmodified POR products
    Douglas Marchuk, Mitchell Drumm, Ann Saulino and Francis S.Collins
    Howard Hughes Medical Institute and Departments of Human Genetics and Internal Medicine,
    University of Michigan Medical Center, Ann Arbor, Ml 4810-0650, USA

    Submitted December 19, 1990

    Although the polymerase chain reaction (1, 2) (PCR) can be used plasmid allows the blue versus white color selection for insert
    to produce a large amount of a specific DNA from a complex containing clones, since a small percentage of vector molecules
    source, cloning the PCR products has not proven to be escape thymidine addition. Sequencing of the insert containing
    straightforward. Restriction endonuclease sites are often clones reveals the insertion of a single T:A base pair between
    incorporated into the oligonucleotide primers used for the EcoRV site and the PCR primer sequence. This procedure
    amplification, so that cleavage of the product will create sticky is at last one hundred fold more efficient that attempting to clone
    ends that can theoretically be ligated to an equivalently cut vector the PCR products into a blunt-ended cut vector.
    (3). Unfortunately, many restriction endonucleases fail to cleave The 3' overhangs of PCR products have been exploited in a
    when their recognition sequences are located within a few base commercially prepared cloning system (TA Cloning kit of
    pairs of the end of a DNA fragment (4, 5). Ligation-independent Invitrogen, San Diego, CA). However, the simple procedure
    PCR cloning schemes (6, 7, 8) involve the addition of at least outlined here can be readily adapted to any plasmid or viral DNA
    12 bases to the 5' end of the primer, which can increase the cost based vector with a unique blunt-ended restriction endonuclease
    of synthesis substantially when done routinely. Attempts to clone site. The procedure is very general so that a single batch of
    PCR products as blunt-ended fragments have been very prepared vector can be used for cloning any PCR product without
    inefficient, due to the template-independent terminal transferase having to redesign oligonucleotide primers. Nine of nine different
    activity of Taq polymerase, which results in the addition of a PCR products were cloned into a plasmid T-vector on the first
    single nucleotide at the 3' end of the fragment (9, 10). This attempt. Additionally, it will find more general use in cloning
    nucleotide is almost exclusively an adenosine, due to the strong schemes where two DNA fragments with incompatible sites need
    preference of the polymerase for dATP (9). Thus, cloning the to be joined together. Fragments can be made blunt-ended by
    products as blunt-ended fragments requires enzymatic processing filling in using Klenow (5' overhang) or trimmed with T4 DNA
    to remove of the 3' overhang using an enzyme with 3' to 5' polymerase (3' overhang). One fragment can subsequently be
    exonuclease activity (11). treated with Taq polymerase in the presence of dATP, the other
    This template-independent activity of Taq polymerase can be in the presence of dTTP, creating complementary one base pair
    exploited to create a cloning scheme which has the efficiency sticky ends for more efficient ligation.
    of sticky end cloning, but requires no additional enzymatic
    modification of the PCR product. Bluescript (Stratagene, La Jolla,
    CA) plasmid is digested with EcoRV, and incubated with Taq
    polymerase (1 unit/ ,ug plasmid/20 ,Il volume) using standard
    buffer conditions (50 mM KCl, lOmM Tris pH 8.3, 1.5 mM REFERENCES
    MgCl2, and 200 jig/ml BSA) in the presence of 2 mM dTTP 1. Saiki,R.K., Scharf,S., Faloona,F., Mullis,K.B., Hom,G.T., Erlich,H.A.
    for 2 hours at 70°C. The absence of any other nucleotides in and Arnheim,N. (1985) Science 230, 1350-1354.
    the reaction results in the addition of a single thymidine at the 2. Saiki,R.K., Celfand,D.H., Stoffel,S. Scharf,S.J., Higuchi,R., Hom,G.T.,
    3' end of each fragment. After phenol extraction and Mulfis,K.B. and Erlich,H.A. (1988) Science 239, 4870-4891.
    precipitation, the T-vector is ready for cloning. PCR products 3. Scharf,S.J., Horn,G.T. and Erlich,H.A. (1986) Science 233, 1076-1078.
    are gel purified to avoid cloning spurious bands, and ligated to 4. Crouse,J. and Amorse,D. (1986) Focus 8, 9.
    5. Kaufman,D.L. and Evans,G.A. (1990) BioTechniques 9, 304-306.
    the vector at 14°C. The vector and PCR products have 6. Shuldiner,A.R., Scott,L.A. and Roth,J. (1990) Nucl. Acids Res. 18, 1920.
    complementary single base 3' overhangs. Vector self-ligation 7. Jones,D.H., Sakamoto,K., Vorce,R.L. and Howard,B.H. (1990) Nature 344,
    events are prohibited by the 3' thymidine overhang, and 793-794.
    concatamerization of the insert is prohibited by the 8. Aslanidis,C. and de Jong,P.J. (1990) Nucl. Acids Res. 18, 6069-6074.
    9. Clark,J.M. (1988) Nucl. Acids Res. 16, 9677-9686.
    unphosphorylated 5' end, contributed by the oligonucleotide 10. Mole,S.E., Iggo,R.D. and Lane,D.P. (1989) Nucl. Acids Res. 17, 3319.
    primer, as well as the 3' adenosine overhang added by Taq 11. Henrsley,A., Amheim,N., Toney,M.D., Cortpssi,G. and Oalas,D.J. (1989)
    polymerase during the PCR reaction. The use of the Bluescript Nucl. Acids Res, 17, 6545-655 1.

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